Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that resembles physiological aging but presents in early childhood. Understanding the mechanism by which HGPS triggers aging may reveal novel aspects of normal aging. HGPS is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins associated with the inner nuclear membrane. The mutations increase use of a cryptic splice site and the deletion of a proteolytic processing site resulting in the abnormal accumulation of farnesylated prelamin A variant called progerin in the nuclear lamina. Mutations in the ZMPSTE24 gene encoding the protease that processes prelamin A result in a similar accumulation of farnesylated prelamin A and cause restrictive dermopathy (RD), a related but more severe progeriod disease. Low levels of progerin have also been detected in normal cells where they may contribute to aged phenotypes. Although morphological alterations in the nuclear envelope have been described in HGPS, RD and normal aging, the mechanism by which progerin leads to abnormal cellular function and disease is unknown. It is likely that progerin expression alters the nuclear lamina and that this affects other structures in the cell. An emerging body of evidence has suggested that the nucleus is connected to the cytoskeleton via a complex of interacting proteins known as the LINC complex. These proteins include the lamins, Suns and nesprin proteins. Suns are transmembrane proteins in the inner nuclear membrane that interact directly with lamins and nesprins. Nesprins are in the outer nuclear membrane and associate with Suns and cytoskeletal elements such as actin. We hypothesize that progerin expression leads to a disruption in the LINC complex, resulting in defective nucleocytoskeletal connections and consequently alterations in nuclear movement and centrosome positioning. Using a model system of fibroblasts undergoing polarization, we have obtained preliminary data showing that expression of progerin alters localization of nesprin2G in the nucleus and interferes with nuclear movement and centrosome positioning. In Aim1 of the proposed experiments, we will test whether farnesylated progerin and prelamin A interfere with LINC complex formation and function and identify where in the pathways for nuclear movement and centrosome positioning progerin acts. In Aim 2, we will determine how progerin affects the biophysical behavior of LINC complex components by examining their mobility in the nuclear membranes and their interactions with each other. In Aim 3, we will test whether progerin is an important component of normal aging by examining whether normal aged cells exhibit defects in the LINC complex and whether the defects can be ameliorated by interventions that reduce progerin expression or farnesylation. These studies will establish whether defects in nucleocytoplasmic interactions contribute to cellular dysfunction in progeria and in normal aging. PUBLIC HEALTH RELEVANCE: An abnormal form of the nuclear protein lamin A is expressed at high levels in cells from patients with the Hutchinson-Gilford progeria syndrome and at low levels in normal aged cells. How the abnormal lamin A affects cell function and aging is unknown. We will explore whether the abnormal lamin A alters proteins in the nuclear membrane that mediate the attachment of the nucleus to cytoskeletal filaments in the cell's cytoplasm, thereby causing defects in cell migration and polarity that could underlie aging.